There are numerous applications where 3-D imaging of objects is desirable. Applications include the electromagnetic (EM) spectrum to ultrasonic frequencies. Instruments for example include Radio Frequency (RF) radar, optical radar, X-ray imagers, optical microscopes, and ultrasound. In general, the wave properties of the probing signal in combination with the material properties of the test object limits the 3-D resolution of the imager. In classical wave optics (EM radiation), wave diffraction sets a limit on the smallest size the optical imager can see in the transverse direction, this limit called the Abbe limit given by 0.5λ/N.A., where N.A is the numerical aperture of the aperture times the refractive index n of the medium in which the object is observed.
For a given microscope objective lens, N.A.=n sin θ, where θ is the maximum half angle that the lens can capture to pick up the highest spatial frequency in the object. In the axial direction, diffraction also plays its limiting role such as in confocal microscopes where the axial resolution (Full-Width-Half-Max: FWHM) can be typical given by 1.4nλ/(N.A.)2. For example, with n=1 (air), N.A.=1.3, and red light with λ=633 nm, the transverse and full-width-half-max axial resolution are 0.24 microns and 0.5 microns, respectively. In alternate Optical Coherence Tomography (OCT) systems, the axial resolution is limited by the coherence length limit of the illuminating radiation, i.e., broader the EM bandwidth, the smaller the axial resolution. In effect, both axial and transverse resolution of optical imagers is on the order of the optical wavelength. It would be highly desirable if optical imagers could see in 3-D with resolution far better than the order of wavelength, perhaps, a 100 or 1000 times smaller than the wavelength. Ultimately, the voxel produced would be a high resolution voxel no longer limited in size by the classic diffraction or coherence limits.
Today, the Abbe resolution still holds strong in optical imagers/microscopes. Various approaches have been tried to improve the axial and transverse resolution of microscopes. One way to get better resolution is via image processing algorithms. Images can get blurred due to fast motion of objects and/or because the image is out of focus because of imperfect imaging conditions. Image sharpening algorithms have been developed that can reduce the image blur or defocus giving higher resolution images. One such successful algorithm involves taking high resolution shifts of low resolution images and then Iterative Back-Projection (IBP) processing to generate the high resolution image. Such an iterative algorithm has been applied to produce higher resolution images such as in remote sensing, and 2-D and 3-D super-resolution on single shot Magnetic Resonance Imaging (MRI). Other image restoration algorithms have also been applied to 3-D optical microscopy to improve imaging resolution. Here, complete 2-D image slices are acquired for different finely tuned focus positions and then this image data is processed. Other successful image processing algorithms applied to high resolution imaging via use of shifted low resolution images include Maximum Likely-Hood Estimation and Iterative Blind Deconvolution.
In these computer based algorithms, one uses the given (by theory) or acquired (by experiment) or estimated Point Spread Function for the imager along-with the high resolution shifted images to reconstruct the high resolution image or 3-D data. As an example, given a low resolution voxel size of X per side, a high resolution shift would be of X/(2N) implemented for both image directions and 2N×2N images are acquired and then processed to finally give a high resolution image with a pixel size of X/N per side. In effect, using these computer algorithms as is done today, massive amount of data processing is required.
Unlike the prior art, the present invention 3-D imaging scheme uses line or one-dimensional (1-D) signal space data along the axial direction to produce the needed high resolution 1-D slices instead of the much larger image or two-dimensional (2-D) signal space data acquired in the transverse direction suffering the Abbe limit.
In addition, prior art shows that breaking the optical limits is not a repudiation of the laws of physics. Nature performs similar interpolation-like brain processing when human vernier acuity tasks are performed to give an order of magnitude better vision than the eye spatial sampling hardware (i.e., cone spacing) allows. Analysis of this phenomenon with broadly tuned sensors yields very high spatial resolutions (error less than 10−4). This interpolation technique of reconstructing fine detail from coarse input sensors can also be utilized to enhance biological and artificial spectrum analyzers.
Cytology, the study of cells using a microscope can greatly benefit from the present inventions sub-wavelength beyond Abbe diffraction limit 3-D imaging as many biological structures/organelles/molecules have diameters in the nanometers scales, e.g., Golgi vesicles at 50 nm, microtubules (25 nm), antibodies (10-15 nm), nuclear pore (9-12 nm), fluorescent proteins/GFP (4-5 nm), cytochrome c (3 nm), and collagen molecule (1.5 nm). In addition, today nanotechnology is offering great promise in medicine such as via quantum dots, nanoshells, and nano-particles that require nano-scale 3-D optical imaging for tissue targeting, nano-medicine delivery, and tissue diagnostics.
It is well known that optical microscopes are a very versatile tool used in a variety of scientific and engineering disciplines ranging from biomedical imaging to electronic and optical chip inspection. Compared to electron microscopy where special sample preparation is required, optical techniques are particularly non-invasive when live tissue cannot be prepared due to material distortions arising from certain treatments. Today, light microscopy can deliver nano-scale readings using optical interference and signal processing techniques. The visible light spectrum is a dominant spectrum for optical microscopy and applies to numerous materials under inspection, screening, diagnosis, and measurement and hence is the subject of the proposed program.
One very important function of optical microscopes is material birefringence measurements where the material ordinary and extraordinary indices are determined indicating the polarization sensitivity of the material. Birefringence meters are used in applications such as characterization of thin films, laser crystals and plate glass for liquid crystal displays—and general quality control metrology. In addition, the medical imaging community also benefits from birefringence measurements of biological tissue where, for example, healthy liver tissue has been shown to have isotropic structure while unhealthy tissue exhibits anisotropic structures or birefringence. In addition, forms of skin cancer have also shown embedded birefringence.
Today, a custom designed heterodyne polarization microscope produces the most accurate (e.g., ±0.008 nm retardation repeatability) birefringence data, such as by the Exicor meter by Hinds Instruments. The Exicor/Hinds Model AT birefringence meter offers the best performance in optical retardation measurements. The basic system uses a 50 KHz Photo-Elastic Modulator (PEM) in combination with polarizer and analyzer optics to produce the optical phase and amplitude data for test material birefringence calculation as described in B. Wang, T. Oakberg, “A New Instrument for Measuring Both the Magnitude and Angle of Low-level Birefringence,” Rev. Sci. Instrum., Vol. 70 Issue 10, 3847-3854, October 1999. The strength of this system comes from its in-line interferometric design coupled with heterodyne optical detection at an RF of 50 KHz leading to low 1/f (f: frequency) noise in the detection electronics. At the red 633 nm wavelength, it measures retardation over a range between 0.005 nm to 100 nm. The standard model is designed to measure birefringence at one wavelength. With an option, the same instrument can be upgraded to measure retardation and three distinct wavelengths of 436 nm, 546 nm, 633 nm, thus provide some spectral information about the sample under test. The instrument cannot function as a profilometer, a 3-D imaging confocal microscope, or a wide spectral range tunable instrument.
A second important function of microscopes is profilometry where surface deformations is optically measured to implement Non-Destructive Testing (NDT) in electronic semiconductor processing, optical chip fabrication, MEMS device inspection, photovoltaic solar panels assembly, liquid crystal display production, and data storage systems. These best profilometry systems such as from Zygo Corp. use Scanning White Light Interferometry (SWLI) with profile resolutions less than 1 nm and range of 0.15 mm for an optimized model.
The present invention pursues a multi-function instrument that realizes a profilometer with equivalent or better performance of a Zygo scanning white light interferometry system. Profilometry using optics has deployed various methods such as chromatic aberration lens based profile coding and the most dominant approach, white light interferometry [see Leslie Deck and Peter de Groot, “High-speed noncontact profiler based on scanning white-light interferometry,” Appl. Opt. 33, 7334-7338 (1994) and Peter De Groot, Xavier Colonna de Lega, Jan Liesener, and Michael Darwin, “Metrology of optically-unresolved features using interferometric surface profiling and RCWA modeling,” Opt. Express 16, 3970-3975 (2008)].
Today, two of the most advanced profilometers are from Zygo Corporation. One profilometer uses Phase Shift Interferometry (PSI) with near 1 nm height resolution over a 138 nm height while and another uses scanning white light interferometry system with a typical range of 0.15 mm and 20 mm for an advanced version. Motion of the test sample or interferometer head in the axial direction is used to record white light interferograms that are processed by image processing algorithms to produce the profile map. The problem is, the dominant scanning white light interferometry system profilometer is not designed for microscope confocal imaging or birefringence measurements or agile spectroscopy.
A third important function of microscopes is confocal microscopy where three dimensional optical imaging of a test sample is realized. A dominant application of confocal microscopy, more specifically, Laser Scanning Confocal Microscopy (LSCM) is biological imaging. A particular application for laser scanning confocal microscopy is implementing fluorescence-based imaging and here the confocal method has shown repeatable results with 200 nm lateral resolution and 500 nm axial resolutions. Hence, the laser scanning confocal microscopy is considered a vital tool in neurobiology, physiology, development biology, and cell and molecular biology. Typical diameter dimensions of biological specimens such as animal cells range between 10-30 micron, cell nucleus range between 3-10 micron, lyosomes between 200-500 nm, hemoglobins around 30 nm, and Collagen molecule around 1.5 nm.
Carl Zeiss Model LSM 710 sets the performance standards for a laser scanning confocal microscopy. Over the years, Carl Zeiss is internationally recognized as a leader in laser scanning confocal microscopy via for example the Zeiss Model 710 laser scanning confocal microscopy. The key points to note here is that axial direction scanning is done by a mechanical motion of either the objective or the sample stage using piezo-actuators with typically 30 nm resolution. Because biological imaging, in particular, in-vivo or live samples can be perturbed by the slightest motion of sample or fluid (i.e., index matching fluid) associated with a high NA: Numerical Aperture objective, ideally one would like to avoid any sample-coupled motion. For example, this mechanical motion based effect can be pronounced in applications such as imaging of non-adherent cells in free floating media and high magnification patch clamp work in neuroscience. Presently, the Zeiss instrument cannot avoid this design limitation associated with sample/objective motion. Also note that the exact laser scanning confocal microscopy performance depends on the specific high N.A. objective used, the laser wavelength, the scan mechanics and its scan steps and modes. A typical performance for visible light laser scanning confocal microscopy is 200 nm lateral resolution, 500 nm axial resolution, and less than 1 second acquisition time per 512×512 pixels image. Again, the dominant laser scanning confocal microscopy is not designed for birefringence measurements and extended range of greater than 500 micron profilometery.
A fourth important function of microscopes is spectral microscopy where optical imaging on a wavelength basis of a test sample is realized. Typically, spectral imaging in a laser scanning confocal microscopy is achieved by providing several independent input ports to the system, as is done in the Carl Zeiss Model LSM 710 with separate fiber port inputs. These laser scanning confocal microscopy devices provide excellent spectral and confocal imaging modalities, but are not designed to enable birefringence measurements or extended depths greater than 500 micron profilometry data.
To overcome the problems associated with the prior art, what is needed is methods, systems and instruments that pursue a multi-function instrument that realizes a laser scanning confocal microscopy and spectral imager with better or equivalent performance of a Carl Zeiss SWLI model 710 type system. Furthermore, the spectral imager will need to have the potential to implement broadband light optical coherence tomography (OCT) via a Radio Frequency (RF) domain Fourier Transform (FT) mode.
Recently, there have been efforts to turn single wavelength laser scanning confocal microscopes into spectral microscopes using a broadband white light source and wavelength tuning and/or wavelength sensitive optical detection, e.g., via grating spectrometer such as described in C. Dunsby et al., “An electronically tunable ultrafast laser source applied to fluorescence imaging and fluorescence lifetime imaging microscopy,” J. Phys. D Appl. Phys., 37, 3296-3303 (2004); T. Betz, et al., “Excitation beyond the monochromatic laser limit: simultaneous 3-D confocal and multiphoton microscopy with a tapered fiber as white-light laser source,” J. Biomed. Opt. 10, 054009 (2005); G. McConnell, S. Poland, and J. M. Girkin, “Fast wavelength multiplexing of a white-light supercontinuum using a digital micromirror device for improved three-dimensional fluorescence microscopy,” Rev. Sci. Instrum. 77, 013702 (2006); J. H. Frank, A. D. Elder, J. Swartling, A. R. Venkitaraman, A. D. Jeyasekharan, and C. F. Kaminski, “A white light confocal microscope for spectrally resolved multidimensional imaging” J. Micros. Oxford 227, pp. 203-215 (2007).
Most laser scanning confocal microscopy devices including the high end Carl Zeiss model 710 can function as a spectral microscope since the microscope has several input laser line ports or as the Model 710 design shows, a broadband visible source is coupled to a single acousto-optic tunable filter (AOTF) device to select the wavelength. Again, note that Zeiss spectral microscope is not designed for birefringence measurements and extended range greater than 500 micron profilometery. In addition, at present, these laser scanning confocal microscopy designs cannot implement broadband light optical coherence tomography.